Department of Cell Biology
Medical Subject Headings
Animals; Binding Sites; *Computational Biology; Gene Regulatory Networks; Humans; Mice; *Muscle Development; Muscle Proteins; Mutagenesis, Site-Directed; MyoD Protein; NIH 3T3 Cells; *Promoter Regions, Genetic
BACKGROUND: Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative in silico analysis coupled with an experimental promoter characterisation to delineate these mechanisms.
RESULTS: Analysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in ms1 mRNA. An in silico comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with ms1 promoter/luciferase reporters and myogenic regulatory factor (MRF) over-expression plasmids revealed specific sensitivity of the ms1 promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2) within identified evolutionary conserved regions (ECRs, alpha and beta). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs alpha and beta correlates with the temporal induction in ms1 mRNA.
CONCLUSION: These findings suggest that the tissue specific and differentiation dependent up-regulation in ms1 mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the ms1 5' upstream sequence. We believe, through its activation of ms1, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination, differentiation and regeneration.
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Citation: BMC Mol Biol. 2008 May 19;9:50. doi:10.1186/1471-2199-9-50. © 2008 Ounzain et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Link to article on publisher's site
Ounzain, Samir; Dacwag, Caroline S.; Samani, Nilesh J.; Imbalzano, Anthony N.; and Chong, Nelson W., "Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte stress 1 gene promoter" (2008). Imbalzano Lab. Paper 4.